For decades it has been known that hemocyanins are among the most potent of immunogens. Hemocyanins, including Keyhole Limpet Hemocyanin (KLH; from the Prosobranch Gastropod Mollusc Megathura crenulata) have been widely used and extensively studied. See Haris et al., “Keyhole limpet hemocyanin (KLH): a Biomedical Review,” Micron, 30(6):597-623 (1999). The high antigenicity of purified hemocyanins, coupled with the relative ease of covalent conjugation with other antigens, has historically made KLH and other hemocyanins a common and familiar tool for establishing baseline immune responses in clinical research and as immunogenic carriers of many haptens in biomedical research.
Uses for hemocyanins have recently expanded, as they are now being tested as therapeutic agents and adjuvants. For example, as an active biotherapeutic agent the hemocyanin KLH is currently being tested clinically in the treatment of certain cancers, including superficial transitional cell carcinoma of the bladder (TCC) (Haris et al. (1999), and Swerdlow et al., “Keyhole Limpet Hemocyanin: Structural and Functional Characterization of Two Different Subunits and Multimers,” Comparative Biochem. & Physiology, Part B, Biochem. & Mol. Biol., 113(3):537-48 (March 1998)), metastasis breast cancer (Biomira, Inc. Company Press Release, Biomira.com, 2001), malignant melanoma, and also as an immune response assay in AIDS research (Kahn et al., “A Phase I study of HGP-30, An Amino Synthetic Peptide Analog Sub-Unit Vaccine in Seronegative Subjects,” AIDS Res. Hum. Retrovirus, 8:1321-1325 (1992); and WO 90/03984 for “Human Immunodeficiency Virus (HIV) Proteins and Peptides Containing the Principal Neutralizing Domain and Their Use in Diagnosis, Prophylaxis, or Therapy of AIDS”). Moreover, hemocyancins are a promising tumor vaccine carrier. See e.g., Thurnher et al., “Dendric Cell-Based Immunotherapy on Renal Cell Carcinoma,” Urol. Int., 61:67-71 (1998); Slovin et al., “Peptide and Carboyhydrate Vaccines in Relapsed Prostrate Cancer: Immunogenicity of Synthetic Vaccines in Man,” Cancer Center Sernin Oncol., 26:448-454 (1999); Massaia et al., “Idiotype Vaccination in Human Melanoma: Generation of Tumor-Specific Immune Responses After High-Dose Chemotherapy,” Blood, 94:673-683 (1999); Fujii et al., “Presentation of Tumor Antigens by Phagocytic Dendritic Cell Clusters Generated From Human CD34+ Hemotopoietic Progenitor Cells: Induction of Autologous Cytotoxic T Lymphocytes Against Leukemic Cells in Acute Mylogeneous Leukemia Patients,” Cancer Res., 59:2150-2158 (1999); Ragupathi et al., “Vaccines Prepared With Sialyl-Tn and Sialyl-Tn Trimers Using 4-(4-maleimidomethyl) Cyclohexane-1-Carboxyl Hydratide Linker Group Result in Optimal Antibody Titers Against Ovine Submaxillary Mucin and Sialyl-Tn-Positive Tumor Cells,” Cancer Immunol. Immunother., 48:1-8 (1999); Sloven et al., “Carbohydrate Vaccines in Cancer: Immunogenicity of a Fully Synthetic Globo H Hexasaccharide Conjugate in Man,” PNAS, USA, 96:5710-5715 (1999); Hsu et al., “Tumor-Specific Idiotype Vaccines in the Treatment of Patients with B-Cell Lymphoma—Long Term Results of a Clinical Trial,” Blood, 89:3129-3135 (1999); Dickler et al., “Immunogenicity of a Fucosyl-GM1-Keyhole Limpet Hemocyanin Conjugate Vaccine in Patients with Small Cell Lung Cancer,” Clin. Cancer Res., 5:2773-2779 (1999); Adluri et al., “Specific Analysis of Sera From Breast Cancer Patients Vaccinated with MUC1-KLH Plus QS-21,” Br. J. Cancer, 79:1806-1812 (1999); and Sandmaier et al., “Evidence of a Cellular Immune Response Against Sialyl-Tn in Breast and Ovarian Cancer Patients After High Dose Chemotherapy, Stem Cell Rescue, and Immunization with Theratope STn-KLH Cancer Vaccine,” J. Immunotherapy, 22:55-66 (1999).
Structure of Gastropod Hemocyanins
To date, isolation of hemocyanins from animals is the only source of these proteins, as efforts to recombinantly produce the proteins have not yet succeeded. Hemocyanins are complex proteins. The most complex mulluscan hemocyanin version is found in gastropods. Biologically, hemocyanins from gastropod molluscs (such as KLH and the hemocyanin from Haliiotis tuberculata, HTH) are blue copper proteins which serve as oxygen carriers in the blood of the animal. The gastropod protein is a hollow cylinder of about 35 nm in diameter with an intricate internal structure. This cylinder is a didecamer based on a 400 kDa polypeptide (the subunit) which forms, in an anti-parallel manner, a stable homo-dimer. Five such homo-dimers constitute the basic cylinder (the decamer, molecular mass of about 4 Mda), which pairwise assemble face-to-face to form the quaternary structure usually found in vivo. Markl et al., J. Cancer Res., 127(Suppl. 2):R3-R9 (2001). The gastropod hemocyanin subunit itself is subdivided into eight different functional units (FUs, termed FU-a to FU-h, about 50 kDa each).
Gastropod molluscan hemocyanins occur as two distinct isoforms. Each of these molecules is based on a very large polypeptide chain, the subunit which is folded into a series of eight globular functional units. Twenty copies of this subunit form a cylindrical quaternary structure. Markl et al., “Marine Tumor Vaccine Carriers: Structure of the Molluscan Hemocyanins KLH and HTH,” J. of Cancer Res., 127, Supplm. 2, pp. R309 (October 2001).
The first complete primary structure of a gastropod hemocyanin subunit was described in 2000. The 3404 amino acid sequence of the hemocyanin isoform HTH1 from Haliiotis tuberculata is the largest polypeptide sequence ever obtained for a respiratory protein. Lieb et al., “The Sequence of a Gastropod Hemocyanin (HTH1),” J. of Bio. Chem., 275:5675-5681 (2000). The cDNA comprises 10,758 base pairs and includes the coding regions for a short signal peptide, the eight different functional units, a 3′-untranslated region of 478 base pairs, and a poly(A) tail. Id. Only recently were the genes coding for molluscan hemocyanins described. Lieb et al., “Structures of Two Molluscan Hemocyanin Genes: Significance for Gene Evolution,” PNAS, USA, 98:4546-4551 (Apr. 10, 2001).
Isolation of Hemocyanin
Because hemocyanins cannot yet be made recombinantly, the proteins must be isolated from hemolymph obtained from source animals. Traditionally, hemocyanin was obtained from hemolymph from the Prosobranch Gastropod Mollusc Megathura crenulata. More recently, the market for gastropod hemocyanins has expanded to include hemocyanin from Haliotis tuberculata and Concholepus concholepus. The hemolymph from other gastropod molluscs is also under investigation for useful properties.
There are a variety of well-known methods for purifying hemocyanins from crude hemolymph, which is the biological source of hemocyanins. These methods include differential centrifugation, gel-permeation chromatography, and ion-exchange chromatography. U.S. Pat. No. 5,407,912 to Ebert for “Method of Treating Bladder Cancer with a Keyhole Limpet Hemocyanin.” Purified hemocyanins are commercially available in many forms.
Despite extensive literature regarding methods for purification of hemocyanins, the only methods described for collection of crude hemolymph from the Prosobranch Gastropod source animals to produce commercially valuable quantities of hemolymph require incision of the vascular system causing death of the source animal. Vanderbark et al., “All KLH Preparations Are Not Created Equal,” Cellular Immunology, 60:240-243 (1981). Methods described for collection of hemolymph for research purposes involve inserting a needle into the muscle of the foot to penetrate the pedal blood sinus. Harris et al., “Keyhole Limpet Haemocyanin: Negative Staining in the Presence of Trehalose,” Micron, 26(1):25-33 (1995).
Due to the anatomy of the vascular system of gastropod molluscs, the pedal sinus does not contain a significant volume of hemolymph and is not readily re-supplied with hemolymph from the heart. Additionally, insertion of a needle through the muscle of the foot results in muscular contractions that further restrict blood flow to the pedal sinus. As a result, the described methods for extraction of hemolymph either are inherently lethal, or are sufficient to only to yield minute quantities of hemolymph for research purposes.
Historically, these limitations on the supply of hemocyanins have not been significant because hemocyanins were principally used in research applications requiring only small quantities. More recently however, the incorporation of hemocyanins into promising new therapeutic products (see e.g., Jurincic-Winkler et al., “Antibody Response to Keyhole Limpet Hemocyanin (KLH) Treatment in Patients with Superficial Bladder Carcinoma,” Anticancer Res., 16(4A):2105-10 (1996); and Biomira, Inc. Company Press Release, Biomira.com, 2001) has resulted in the need for a sustainable supply of commercial quantities of hemocyanin produced under conditions that meet the health and safety standards imposed by the United States Food and Drug Administration and other regulatory agencies.
This need for a uniform and sustainable supply of hemocyanin produced under Good Manufacturing Procedures for pharmaceutical applications has created a need for a method to safely and repeatedly extract commercial quantities of hemocyanin from animals grown in a controlled environment. The present invention satisfies these needs.